The cyclophilins are a family of ubiquitous eukaryotic proteins first identified by their high affinity for the immunosuppressant drug, cyclosporin A: Isoforms are targeted to the cytosol, secretory pathway, and mitochondria. All tested isoforms bind cyclosporin A and have in vitro peptidyl-prolyl isomerase activity. Elucidating the normal physiological role of the cyclophilins may help in understanding their role in mediating the immunosuppressant and cytotoxic activities of cyclosporin A. We previously reported the isolation and characterization of a yeast cyclophilin gene, CPR3. This gene is essential for growth on lactate as a sole carbon source at 37 degrees. We have raised antibodies to the gene product and demonstrated that it is located in the mitochondrial matrix. Further, by analysis of cpr3 mutants, we have determined that the primary defect is not in the synthesis of lactate dehydrogenase, the only enzyme solely involved in lactate metabolism, but rather in some aspect of mitochondrial function which regulates synthesis of all cytochromes. Unlike the other cytochrome genes however, the lactate dehydrogenase gene has no basal level of transcription; the specificity of the lactate phenotype is explained by the tight transcriptional regulation of the lactate dehydrogenase gene rather than by any special requirement for the CPR3 gene product in the maturation of lactate dehydrogenase. To define the defect more precisely, we have isolated dominant revertants from cpr3- mutants which are capable of growth on lactate at 37 degrees Genomic fragments from this revertant which complement the defect in the cpr3- mutants have been isolated and are being analyzed.